Elucidation of the mescaline biosynthetic pathway in peyote (Lophophora williamsii).

Peyote (Lophophora williamsii) is an entheogenic and medicinal cactus native to the Chihuahuan desert. The psychoactive and hallucinogenic properties of peyote are principally attributed to the phenethylamine derivative mescaline. Despite the isolation of mescaline from peyote over 120 years ago, the biosynthetic pathway in the plant has remained undiscovered. Here, we use a transcriptomics and homology-guided gene discovery strategy to elucidate a near-complete biosynthetic pathway from l-tyrosine to mescaline. We identified a cytochrome P450 that catalyzes the 3-hydroxylation of l-tyrosine to l-DOPA, a tyrosine/DOPA decarboxylase yielding dopamine, and four substrate-specific and regiospecific substituted phenethylamine O-methyltransferases. Biochemical assays with recombinant enzymes or functional analyses performed by feeding putative precursors to engineered yeast (Saccharomyces cerevisiae) strains expressing candidate peyote biosynthetic genes were used to determine substrate specificity, which served as the basis for pathway elucidation. Additionally, an N-methyltransferase displaying broad substrate specificity and leading to the production of N-methylated phenethylamine derivatives was identified, which could also function as an early step in the biosynthesis of tetrahydroisoquinoline alkaloids in peyote.

Detection of mescaline in human hair samples by UPLC-MS/MS: Application to 19 authentic forensic cases.

Mescaline, a natural alkaloid found in the peyote cactus (Lophophora williamsii) in the Americas, has gradually become a drug of abuse in China because of its psychedelic properties. Its intake may lead to hallucinations and confusion or even be life-threatening. Mescaline is classified as a class Ⅰ psychotropic drug in China, which means its use in medicine or scientific research is under strict control of the government. However, studies on surveillance of mescaline abuse in the Chinese population are lacking. A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination and quantification of mescaline in hair. The method had good linearity in the range from 10 to 1000 pg/mg, with the limit of detection (LOD) of 3 pg/mg and the limit of quantitation (LOQ) of 10 pg/mg. The total runtime was 5 min. Acceptable intraday and interday precision (RSD < 15%) and accuracy (bias, -11.2% ∼ 6.8%) were achieved. The recovery was 85.0-101.0%, and the matrix effect was 92.0-105.0%. The validated method was successfully applied to 19 real forensic cases. The concentrations of mescaline in hair ranged from 10 to 784 pg/mg. The method has the benefits of simple sample preparation, high sensitivity, and short running time, making it suitable for large-scale quantitative surveillance analysis of mescaline in forensic toxicology.

Single Nucleotide Polymorphism Assay for Genetic Identification of Lophophora williamsii.

Lophophora is a member of the Cactaceae family, which contains two species: Lophophora williamsii and L. diffusa. Lophophora williamsii is an illegal plant containing mescaline, a hallucinogenic alkaloid. In this study, a novel method based on a single nucleotide polymorphism (SNP) assay was developed for identifying L. williamsii; this assay reliably detects SNPs within chloroplast DNA (rbcL, matK, and trnL-trnF IGS) and was validated for identifying Lophophora and L. williamsii simultaneously. The chloroplast DNA sequences from four L. williamsii and three L. diffusa plants were obtained and compared using DNA sequence data from approximately 300 other Cactaceae species available in GenBank. From this sequence data, a total of seven SNPs were determined to be suitable for identifying L. williamsii. A multiplex assay was constructed using the ABI PRISM® SNaPshot™ Multiplex Kit (Applied Biosystems, Forster City, CA) to analyze species-specific SNPs. Using this multiplex assay, we clearly distinguished the Lophophora among 19 species in the Cactaceae family. Additionally, L. williamsii was distinguished from L. diffusa. These results suggest that the newly developed assay may help resolve crimes related to illegal distribution and use. This multiplex assay will be useful for the genetic identification of L. williamsii and can complement conventional methods of detecting mescaline.

An Efficient Ambient Ionization Mass Spectrometric Approach to Detection and Quantification of the Mescaline Content of Commonly Abused Cacti from the Echinopsis Genus.

Unregulated cacti from the genus Echinopsis are used recreationally as mescaline-containing alternatives to the outlawed peyote. Echinopsis-derived plant materials appear in a variety of nondescript forms, making rapid assessment of whether they are mescaline-containing materials or simply innocuous plant-derived food products, very challenging. Reported here is a DART-HRMS approach for the rapid detection of mescaline in whole plant material and a validated method for the quantification of mescaline in cactus tissue, using mescaline-d9 as the internal standard. Calibration curves exhibited R2 values of ≥0.995, and the method exhibited a LLOQ and a linear range of 1 ppm and 1-100 ppm, respectively. Application of the method to commercially available Echinopsis spp. yielded results consistent with previous studies performed by GC- and LC-MS, with mescaline levels of <2% dry weight in all cases. Therefore, DART-HRMS is a suitable technique for the rapid screening of mescaline and its subsequent quantification within complex plant-derived matrices.

Elucidating the Distribution of Plant Metabolites from Native Tissues with Laser Desorption Low-Temperature Plasma Mass Spectrometry Imaging.

Secondary metabolites of plants have important biological functions, which often depend on their localization in tissues. Ideally, a fresh untreated material should be directly analyzed to obtain a realistic view of the true sample chemistry. Therefore, there is a large interest for ambient mass-spectrometry-based imaging (MSI) methods. Our aim was to simplify this technology and to find an optimal combination of desorption/ionization principles for a fast ambient MSI of macroscopic plant samples. We coupled a 405 nm continuous wave (CW) ultraviolet (UV) diode laser to a three-dimensionally (3D) printed low-temperature plasma (LTP) probe. By moving the sample with a RepRap-based sampling stage, we could perform imaging of samples up to 16 × 16 cm2. We demonstrate the system performance by mapping mescaline in a San Pedro cactus ( Echinopsis pachanoi) cross section, tropane alkaloids in jimsonweed ( Datura stramonium) fruits and seeds, and nicotine in tobacco ( Nicotiana tabacum) seedlings. In all cases, the anatomical regions of enriched compound concentrations were correctly depicted. The modular design of the laser desorption (LD)-LTP MSI platform, which is mainly assembled from commercial and 3D-printed components, facilitates its adoption by other research groups. The use of the CW-UV laser for desorption enables fast imaging measurements. A complete tobacco seedling with an image size of 9.2 × 15.0 mm2 was analyzed at a pixel size of 100 × 100 µm2 (14 043 mass scans), in less than 2 h. Natural products can be measured directly from native tissues, which inspires a broad use of LD-LTP MSI in plant chemistry studies.

GC-MS and GC-MS/MS in PCI mode determination of mescaline in peyote tea and in biological matrices.

Peyote, a cactus containing the hallucinogen mescaline, is used to induce altered states of consciousness in religious ceremonies or for recreational purpose. This study reports a case of an underage boy suspected of mescaline abuse. For this purpose, we analyzed a dark green liquid sample found in the bedroom of the boy whose urine and hair samples were collected shortly after the drink was found. A method by gas chromatography-mass spectrometry tandem mass spectrometry (GC-MS/MS) in positive chemical ionization mode was developed and validated in terms of linearity, specificity, accuracy, and sensitivity for mescaline determination at the low concentrations present in hair. GC-MS analysis of the liquid identified mescaline, while urine was negative; GC-MS/MS segmental hair analysis identified mescaline in the proximal segment (root to 2 cm), while the distal segments were negative. Although peyote was uncommonly encountered, its use was confirmed by segmental hair analysis that can provide long-term information about drugs use.

Rapeseed or linseed in grass-based diets: effects on conjugated linoleic and conjugated linolenic acid isomers in milk fat from Holstein cows over 2 consecutive lactations.

Changes in the distribution of conjugated linoleic (CLA) and conjugated linolenic (CLnA) acid isomers in milk from Holstein cows in response to 4 different oilseed supplements rich in either cis-9 18:1 or 18:3n-3 were determined over 2 consecutive lactations in 58 and 35 cows during the first and second years, respectively. For the first 5 wk of the first lactation, all cows were fed the same diet. Thereafter, cows received 1 of 5 treatments for 2 consecutive lactations, including the prepartum period. Treatments comprised the basal diet with no additional lipid, or supplements of extruded linseeds (EL), extruded rapeseeds (ER), cold-pressed fat-rich rapeseed meal, or whole unprocessed rapeseeds to provide 2.5 to 3.0% of additional oil in diet dry matter. During indoor periods, cows were housed and received a mixture (3:1, wt/wt) of grass silage and hay, whereas cows were at pasture during outdoor periods. Over the entire study, EL resulted in the enrichment of ∆11,13 CLA, ∆12,14 CLA, trans-9,trans-11 CLA, trans-13,trans-15 CLA, ∆9,11,15 CLnA, and cis-9,trans-11,trans-13 CLnA (identified for the first time in bovine milk fat) in milk fat, whereas ER and cold-pressed fat-rich rapeseed meal in particular, increased milk fat trans-7,cis-9 CLA concentration. With the exception of the first indoor period, whole unprocessed rapeseeds decreased cis-9,trans-11 CLA, trans-9,cis-11 CLA, and trans-10,trans-12 CLA abundance. During the second indoor period, EL increased milk trans-9,cis-11 CLA and trans-10,cis-12 CLA concentrations, but the increases in cis-9,trans-11 CLA, cis-12,trans-14 CLA, trans-11,cis-13 CLA, and cis-9,trans-11,cis-15 CLnA concentrations to EL and ER were lower for the second than first indoor period. In contrast to the indoor periods, EL and ER decreased milk cis-9,trans-11 CLA, trans-9,cis-11 CLA, and trans-10,cis-12 CLA concentrations at pasture. The extent of changes in the relative distribution and abundance of CLA and CLnA isomers in milk fat were related to the nature (rapeseed or linseed) and form of oilseed (extruded, cold-pressed fat-rich meal or whole unprocessed) supplement and their interactions with the composition of the basal diet (conserved grass or pasture and dietary starch content). Furthermore, milk fat CLA and CLnA responses to treatments were repeatable between both outdoor periods. Variations in milk fat content and yield measured during the entire study were significantly and inversely associated with milk trans-10 18:1, trans-10,cis-12 CLA, and in particular, trans-9,cis-11 CLA concentrations.

Purification of pharmaceutical preparations using thin-layer chromatography to obtain mass spectra with Direct Analysis in Real Time and accurate mass spectrometry.

Forensic analysis of pharmaceutical preparations requires a comparative analysis with a standard of the suspected drug in order to identify the active ingredient. Purchasing analytical standards can be expensive or unattainable from the drug manufacturers. Direct Analysis in Real Time (DART™) is a novel, ambient ionization technique, typically coupled with a JEOL AccuTOF™ (accurate mass) mass spectrometer. While a fast and easy technique to perform, a drawback of using DART™ is the lack of component separation of mixtures prior to ionization. Various in-house pharmaceutical preparations were purified using thin-layer chromatography (TLC) and mass spectra were subsequently obtained using the AccuTOF™- DART™ technique. Utilizing TLC prior to sample introduction provides a simple, low-cost solution to acquiring mass spectra of the purified preparation. Each spectrum was compared against an in-house molecular formula list to confirm the accurate mass elemental compositions. Spectra of purified ingredients of known pharmaceuticals were added to an in-house library for use as comparators for casework samples. Resolving isomers from one another can be accomplished using collision-induced dissociation after ionization. Challenges arose when the pharmaceutical preparation required an optimized TLC solvent to achieve proper separation and purity of the standard. Purified spectra were obtained for 91 preparations and included in an in-house drug standard library. Primary standards would only need to be purchased when pharmaceutical preparations not previously encountered are submitted for comparative analysis. TLC prior to DART™ analysis demonstrates a time efficient and cost saving technique for the forensic drug analysis community. Copyright © 2011 John Wiley & Sons, Ltd.

New mescaline concentrations from 14 taxa/cultivars of Echinopsis spp. (Cactaceae) ("San Pedro") and their relevance to shamanic practice.

AIM OF THE STUDY: The aim of the present study is to determine in a procedurally uniform manner the mescaline concentrations in stem tissue of 14 taxa/cultivars of the subgenus Trichocereus of the genus Echinopsis (Cactaceae) and to evaluate the relationship (if any) between mescaline concentration and actual shamanic use of these plants. MATERIALS AND METHODS: Columnar cacti of the genus Echinopsis, some of which are used for diagnostic and therapeutic purposes by South American shamans in traditional medicine, were selected for analysis because they were vegetative clones of plants of documented geographic origin and/or because they were known to be used by practitioners of shamanism. Mescaline content of the cortical stem chlorenchyma of each cactus was determined by Soxhlet extraction with methanol, followed by acid-base extraction with water and dichloromethane, and high-pressure liquid chromatography (HPLC). RESULTS: By virtue of the consistent analytical procedures used, comparable alkaloid concentrations were obtained that facilitated the ranking of the various selected species and cultivars of Echinopsis, all of which exhibited positive mescaline contents. The range of mescaline concentrations across the 14 taxa/cultivars spanned two orders of magnitude, from 0.053% to 4.7% by dry weight. CONCLUSIONS: The mescaline concentrations reported here largely support the hypothesis that plants with the highest mescaline concentrations - particularly E. pachanoi from Peru - are most associated with documented shamanic use.

Prehistoric peyote use: alkaloid analysis and radiocarbon dating of archaeological specimens of Lophophora from Texas.

Two archaeological specimens of peyote buttons, i.e. dried tops of the cactus Lophophora williamsii (Lem.) Coulter, from the collection of the Witte Museum in San Antonio, was subjected to radiocarbon dating and alkaloid analysis. The samples were presumably found in Shumla Cave No. 5 on the Rio Grande, Texas. Radiocarbon dating shows that the calibrated 14C age of the weighted mean of the two individual dated samples corresponds to the calendric time interval 3780-3660 BC (one sigma significance). Alkaloid extraction yielded approximately 2% of alkaloids. Analysis with thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) led to the identification of mescaline in both samples. No other peyote alkaloids could be identified. The two peyote samples appear to be the oldest plant drug ever to yield a major bioactive compound upon chemical analysis. The identification of mescaline strengthens the evidence that native North Americans recognized the psychotropic properties of peyote as long as 5700 years ago.

The analysis and distribution of mescaline in postmortem tissues.

Mescaline (3,4,5-trimethoxyphenethylamine) is a hallucinogenic alkaloid found in the peyote cactus. This report documents mescaline distribution in a death caused by multiple gunshot wounds. Mescaline was extracted with a butyl chloride liquid-liquid method and identified by mass spectrometry. Quantitative analysis was performed by gas chromatography using a nitrogen-phosphorus detector. Concentrations of the drug were 2.95 mg/L, 2.36 mg/L, 8.2 mg/kg, and 2.2 mg/kg in blood, vitreous, liver, and brain, respectively.

[Some analytical aspects of mescaline]. / Quelques aspects analytiques de la mescaline.

Mescaline was extracted from a vegetal powder, seazed on the "Côte d'Azur", then analyzed by several techniques (thin layer chromatography, infra-red spectrometry, gas chromatography/mass spectrometry) and determined by high performance liquid chromatography with methyl-amphetamine as internal standard. The powder contained 0.76% of mescaline. The presence of a possible isomer was noted in the powder as well as in a "old" sample of mescaline.

A mescaline associated fatality.

The death of an individual under the influence of mescaline is presented. Concentrations of the drug were 9.7, 70.8, and 1163 micrograms/mL or micrograms/g in blood, liver, and urine respectively.

[Estimation of mescaline and pellotine in Lophophora coulter plants (Cactaceae) by means of the oscillographic polarography]. / Opredelenie meskalina i pellotina v rasteniiakh poda Lornornoiaa coulter (cem. Cactaceae) pri pomoshchi ostsillograficheskoi poliarografii.

Oscillographic polarography has been applied for the mescaline and pellotine estimation. These alkaloids produce in 0.5 N NaOH electrolyte a sharp peak within the cathode region of the oscillogram, each of them showing different potential. It makes possible to estimate them at a concentration of 5.10(-6) g/ml. All the forms of Lophophora williamsii were found to contain mescaline and lower content of pellothine, L. jourdaniana--to have equal content of both alkaloide, L. diffusa and L. fricii--to contain pellotine and only traces of mescaline. Plants grown in the greenhouse accumulated the same amount of alkaloids as native plants. Grafting on roodstock which does not produce essential amount of the alkaloids, does not affect the ability of Lophophora to synthesize mescaline and pellotine.